Preclinical In Vitro Investigation of MDM2 Inhibition in Combination with Antiangiogenic Therapy for Breast Cancer Treatment
, Khalid Alhazzani 2,†, Ahmed Zuwaiel Alanazi 2, Yasser Alqarni 2, Mohammad M. Algahtani 2, Abdullah S. Alhamed 2, Glowi Alasiri 3, Fahad T. Alotaibi 4, Talha Jawaid 1 and Jehad A. Aldali 5Round 1
Reviewer 1 Report
Dear Authors, kindly improve the figure quality. rest is up to mark.
Author Response
Response: We value the reviewer's feedback. The word has been corrected as requested.
Reviewer 2 Report
Corrections to the Manuscript.
This is an invitro study and nowhere in the “topic” and “abstract” the word “invitro” is mentioned.
Therefore, the title should be changed to “An in vitro investigation of therapeutic significance of chemotherapeutic and antiangiogenic drugs using MCF-7 cells”.
The abstract should also be changed. An abstract should look like below
“Combining antiangiogenic drugs with other chemotherapeutic drugs has been found to produce superior therapeutic outcome and prevent drug resistance in a variety of cancers. Methods: Experimental assays for cell viability, and expression of proteins linked to apoptotic and cell survival processes were employed to evaluate the effectiveness of combination therapy. Results: When compared to controls and monotherapies, the combination treatment of axetinib, an antiangiogenic and idasanutlin, a chemotherapeutic agent demonstrated a substantial decrease in cell viability at lower doses, a significant decrease in migration and cell invasion, and a shift toward early and late apoptosis. The study examined the expression of major apoptotic, metastatic, and angiogenic factors, including as MDM2, P21, BCL-2, BCL-XL, and MMP-9, which have showed differential expression at the protein and mRNA levels after combination. Axitinib and idasanutlin in combination at -------decreased tumorigenesis and migration of MCF-7 cell line when compared to other chemotherapeutic medications. The suggested mechanisms of the combination therapy may be due to promotion of the production of apoptotic markers and reduce antiapoptotic markers. Conclusions: Treatments with axitinib and idasanutlin demonstrated effective therapeutic targeting of the primary angiogenic growth factor, and consequently the proastatic arbitrators. The combination therapy will not only eliminate cancer cells, but also inhibit other malignant processes resulting into reduced metastatic cascade.”
Suggested corrections
Put word “gene” between “tumor suppresspr” and “dysregulation” in the sentence “Furthermore, oncogene and tumor suppressor 49 dysregulation are driving forces for many genetic alterations and are considered a major 50 contributor to uncontrolled growth, including the development of breast tumors.”
Define “RCC” in the sentence “Axitinib is an oral, second-generation pan-VEGFR antagonist that binds with 74 high affinity in the kinase domains of VEGFR-1, 2, and 3 and is approved for advanced 75 RCC treatment[9-10]”
Write correctlt the number of cells in the sentence “Microplates 114 (Thermo Scientific, Waltham, Massachusetts, USA) with 1 x 104 cells per well and incu- 115 bated at 37°C under a humidified atmosphere of 5% CO2 for 24 hrs. Then, the medium 116 was removed and replaced by MTT (5 mg/mL) in Phosphate Buffered Saline (PBS) and 117 incubated for 2 to 4 hours at 37°C.”
Since there are a number of Breast Cancer types and treatment optiopn depends on the presence of ER, PR or Her2, the antigenic types of MCF-7 (ER/PR/HER2 status) should be described.
The word “H2O” should be written as “H2O” in the sentence “…(MedChemExpress, USA) into single-stranded complementary DNA (cDNA). Maxima® 148 SYBR Green/Fluorescein qPCR Master Mix, forward and reverse primers, DNA, and 149 RNase-Free H2O (MedChemExpress, USA) were used to determine the BAX, BCL-2, P21, 150 VEGF, TGF-β, MMP-9, and P53”
Change the word “This” into “Reaction” in the sentence “This was amplified for 40 cycles at 95°C for 15 seconds, at 60°C for 30 seconds, and at 72°C for 30 seconds.”
The word “second” should be “secondary” in the sentence “Membranes were incubated with the 175 second antibodies for 2 hours on the shaker at 37°C room temperature then washed.” This sentence should be written as "Membranes were incubated with the secondary antibodies conjugated with Horse reddish peroxidase for 2 hours on the shaker at 37°C room temperature then washed with TBST two times for 10 minutes at room temperature.”
Line 279: The sub-heading “3.6 Effect of axitinib and idasanutlin on BCL-2, BCL-XL, P21, and MDM2 Protein levels 279 in MCF-7 cells.” should be changed to “3.6 Effect of axitinib and idasanutlin on BCL-2, BCL-XL, P21, and MDM2 Protein expression levels in MCF-7 cells.”
Line 305-307: There is no need to write the word “anticancer “ in the sentence “Axitinib anticancer effect inhibited cell proliferation in various cancer types including non-small cell lung cancer [16], prostate [17], and neuroblastoma [18].”
Author Response
Please see the attachment.
Author Response File: 
Author Response.docx
Reviewer 3 Report
The manuscript by Alaseem et al., “Preclinical investigation of MDM2 inhibition in combination with antiangiogenic therapy for breast cancer treatment.”, analyzes the effects of using a combination of axitinib and idasanutlin for the treatment of breast cancer. The authors investigate the ability of a axitinib-idasanutlin combination to inhibit the function of the breast cancer cell line MCF-7. This study looks at the ability of the compounds to promote apoptosis and inhibit cell migration. In addition, the study shows gene expression of BAX, P53, and P21 are increased and BCL-2 is decreased. The authors further show protein expression of BCL-2, BCL-xL and MDM2 is decreased and P21 is increased. Taken together the authors suggest the use of axitinib and idasanutlin in combination inhibited MCF-7 cell growth and migration better than individual treatment. This result is due to induce the expression of apoptotic markes and suress anti-apoptotic markers.
Major Issues:
1. In section 3.2, the authors use the scratch assay to evaluate axitinib and idasanutlin to inhibit MCF-7 cell migration. In the result, the authors indicate a “…reduction of cell migration and invasion compared to control… (line 216-219). The scratch assay is only a measurement of cell migration. Thus, the authors should remove all reference to invasion. If the authors want to include invasion they have to perform an invasion assay (transwell assay, Boyden chamber).
2. Figure 3. The authors show flow cytometry dot plots of cell staining for annexin-V and PI. The authors should include a graph overlay of the early apoptosis, late apoptosis and necrosis for between the 3 treatments. This will provide the reader with a better understand of the results since the authors do not provide a description on how they measured apoptosis and necrosis.
Minor Issues:
1. Figure 2 is missing labels and is incorrectly labeled the images of control, axitinib and idasanutlin are not labeled. The combination 1 label is cut off and indicates ibination 1. This should be fixed
2. Figure 2. The title indicates invasion. The scratch assay only shows migration thus invasion should be removed from the title and the legend.
3. Section 3.3, analyzes annexin-V and PI staining. The authors indicate early apoptosis and late apoptosis (line 238-240). The authors should discuss how they determined early and late apoptosis.
4. In section 3.4 the authors should provide text on the function of BAX. The authors provide the function of P53, P21 and BCL-2 but omit BAX
5. Section 3.5, omit migration and invasion markers from the title. A better title is “ Effects of axitinib and idasanutlin on gene expression of VEGF and bFGF. VEGF and bFGF are well known angiogenic. Although these growth factors have been associated as migration and invasion markers they are not the prototypical markers associated with cancer cell migration and invasion. Thus, I feel indicating VEGF and bFGF are cancer cell migration and invasion markers is overreaching.
Author Response
Please see the attachment.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
Authors have taken the comments seriously and incorporated all suggested modifications.
Author Response
Response: Thank you for taking the time to review our manuscript. We appreciate the valuable insights and suggestions you have provided.
Reviewer 3 Report
The authors do not provide an adequate explanation in the Results on how to interpret Figure 3. They should place the information provided to the reviewer into Section 3.3, line 240 before the sentence, "Axitinib monotherapy...." This will provide the readers an understanding on how to evaluate Figure 3- The top right quadrant represents late apoptotic cells with high levels of Annexin V and PI staining. The bottom right quadrant represents early apoptotic cells with high levels of Annexin V and low levels of PI staining.
Author Response
Response: We appreciate the valuable insights and suggestions you provided. As per your suggestion, we have taken into consideration the comment and made the necessary changes to the manuscript
